PCR is probably the most important contribution among techniques that have revolutionized the uncovering of the human genome. Today, the vast majority of methods for identifying sequences in the human genome involve target sequence amplification through PCR.
A crucial problem with PCR is that when large numbers of specific DNA sequences are simultaneously amplified in the same reaction tube, then undesired amplification products often arise. The undesired amplification products in multiplex PCR are associated with, and increased in relation to, the number of the added primer-pairs. Even with careful attention paid to the design of the primers, PCR is usually limited to 10 simultaneous amplification reactions before false amplification products are formed. Therefore, in research projects that comprise identification and analysis of many nucleic acids sequences, a large number of separate PCRs must be performed.
Today, a PCR generally takes about two hours to perform and requires a defined amount of target material. In investigations where many PCRs must be performed, the projects often prove time consuming, expensive, and require a large collection of target DNA.
Different methods to overcome the problems associated with conventional multiplex PCR have been developed, but none with full success.
PCT publication WO 96/41012 discloses a method for multiplex PCR that entails two rounds of amplification and that uses primer pairs comprising template-specific sequences at their respective 3′ ends and universal primer sequences at their respective 5′ ends. The first round of amplification uses the specific primer sequences and the second amplification uses the universal primer sequences.
DOP-PCR (degenerate oligonucleotide-primed PCR) is a form of PCR which is designed to produce several different products through use of degenerated primers (Zhang, et al. Proc. Natl. Acad. Sci. USA 89, 5847-5851 (1992); Cheung and Nelson Proc. Natl. Acad. Sci. USA 93, 14676-14679 (1996)). The method is mainly used for “whole genome amplification” and lacks the means for selectively choosing a number of targets to be amplified in parallel.
Also, a number of DNA amplification methods that use so called adaptor-ligation PCR have been developed in different formats. Broude, et al., Proc. Natl. Acad. Sci. USA 98, 206-211 (2001) presented an approach to use single specific primers for each target and a single common primer. Kennedy, et al. Nat Biotechnol 21, 1233-1237 (2003) presents a method for fragment selection and complexity reduction through adaptor ligation on a digested whole genome sample. The ligation of adaptors to digested sample is then followed by a PCR that is set to amplified fragments of a certain size. All these methods have in common that they cannot amplify many specific fragments in parallel without amplifying a large collection of undesired DNA targets at the same time. Callow, et al. Nucleic Acids Res 32, E21 (2003) present a technique to use adaptor-ligation PCR together with a specific selection of targets using rounds of Type IIs restriction enzyme cleavage. This method suffers from the lack of ability to amplify a large set of specific targets in parallel, and therefore remains limited in its application.
The method uses Type IIs restriction enzymes that produce 4-base, 5′-overhang of digested genomic DNA to fragment the genome into 32768 variants of overhangs (non-directional). To avoid hybridization and ligation of double-sided adaptors to itself, all 16 palindrome 4-base combinations must be avoided resulting in a design success-rate of 88% for any given Type IIs restriction enzyme. To avoid hybridization and ligation of one double-sided adaptor to another no 4-base overhang combination complementary to another adaptor's overhang can be used. This limitation results in an increasing difficulty of finding suitable adaptors for increasing number of targets to be amplified.
As an example for one round of selection; when a random set of 10 fragments from the human DNA is chosen to be amplified in parallel, the chance of finding all corresponding adaptors is only 50%. Or even worse, when a random set of 50 fragments is chosen to be amplified in parallel, there is almost no chance of finding the pool of corresponding adaptors (2.2.10−9%) that selects all 50 fragments. The limitation of using only 4-base combinations to select all fragments in a complex DNA sample such as the human genome results in that the method can not be used for parallel amplification of large sets of specific targets. The method can be used to amplify subsets of genomes or very few targets in parallel but lacks the freedom of action of amplify large sets of specific targets without producing unwanted DNA.
PCT publications WO 03/012119 and WO 03/044229 disclose methods to specifically circularize genomic fragments and amplify them with so called rolling circle amplification. These two publications do not disclose PCR amplification of the selected fragments and do not contain a description of the design of the selector.
Thus, there exists a need for methods that permit amplification of multiple specific DNA sequences in the same reaction without producing amplification artifacts.